Project Summary/Abstract Nearly 10% of the world has diabetes and this number is projected to increase. Type 2 diabetes occurs when beta cells fail to produce sufficient insulin in the face of insulin resistance. One important aspect of beta cell failure is mitochondrial dysfunction. While mitochondrial have long been known to be critical for glucose stimulated insulin secretion, it is now clear that mitochondria are not simply static metabolic organelles. They are in a constant state of fission and fusion and disruption of this balance affects bioenergetics, mitophagy and cell survival. We propose that mitochondrial morphology could be a novel target for diabetes therapeutics. However, very little is known about how changes in mitochondrial morphology affect beta cell function and survival in vivo. The central objectives of this proposal are to understand how loss of mitochondrial fission affects insulin secretion, beta cell survival and mitophagy. Our specific aims are as follows. Aim 1: Define how mitochondrial fission regulates the metabolic amplifying pathway of insulin secretion. Based on our preliminary data, we hypothesize that mitochondrial fission is required for the NADPH amplifying pathway. We will use optical reporters to ascertain the oscillatory dynamics of NADPH in response to glucose in the setting of acute and chronic Drp1 knockout. Since Drp1 has roles outside of mitochondrial fission, we will use CRISPR interference to silence a pure regulator of mitochondrial fission, Mief1, for comparison with the Drp1 knockout. Finally, we will restore mitochondrial morphology in Drp1 deficient beta cells with concomitant knockdown of the mitochondrial fusion regulator mitofusin-2 (Mfn2) to rescue insulin secretion. Aim 2: Determine the role of mitochondrial fission in beta cell survival and mitochondrial quality control. In non-beta cells, mitochondrial fission is thought to be important for mitophagy but be required for apoptosis. Based on our preliminary data, we hypothesize that loss of Drp1 in the beta cell may protect cells from apoptosis but may?trigger mitophagy and mitochondrial quality control. Aim 3: Establish the relevance of Drp1 in human islets with and without type 2 diabetes. We will knockdown Drp1 in primary human beta cells from non-diabetic patients and measure their oxygen consumption, insulin secretion, and calcium dynamics. To test a role of mitochondrial dynamics in type 2 diabetes pathogenesis, we will measure levels of mitochondrial dynamics proteins in beta cells from type 2 diabetes patients and examine the mitochondrial morphology in these cells. Finally, to test a role of Drp1 downregulation in type 2 diabetes, we will re-express Drp1 in human diabetic beta cells and ask if glucose stimulated insulin secretion can be improved. These studies will lay the foundation for future diabetes therapeutics based on improving mitochondrial dynamics.